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Bioprocessing strategies to improve production of the heterologous protein parathion hydrolase from recombinant Streptomyces lividans were investigated. Initial limitations to increased production were overcome by using large amounts of nutrients and feeding these nutrients throughout the fermentation. Batch addition of such large amounts of nutrients resulted in byproduct acid accumulation. Our data suggest that byproducts resulted from incomplete utilization of peptide medium ingredients and not from an overflow of glucose catabolism. Over extended fed-batch operation, oxygen transfer became limiting and these limitations were overcome by sparging oxygen-enriched gas. When cultivation was continued past about 90 h, we observed that despite nutrient feeding and oxygen enrichment enzyme activities no longer increased. Our results show that during such late cultivation periods the rates of enzyme synthesis and deactivation became balanced. If synthesis is prevented, either by a nutritional limitation or by the addition of the protein synthesis inhibitor chloramphenicol, enzyme activities were observed to decrease. Since deactivation rate constants in these experiments were similar to those observed in cell-free studies, and because extracellular protease activities were not detected in our fermentation, it appears that deactivation results from the inherent instability of the parathion hydrolase enzyme.

A model was formulated to examine specific experimental data of growth and heterologous product formation with recombinant Saccharomyces cerevisiae while incorporating available literature. The model simulated dry cell weight, glucose, ethanol, dissolved oxygen, human Epidermal Growth Factor (hEGF) production, fraction of recombinant cells, oxygen uptake rate, and carbon dioxide production rate for batch, fed batch, and hollow fiber bioreactor configurations. Nineteen differential equations, 24 analytical equations, and 48 parameters were required. Due to the lack of detailed studies needed for the ADH-II and the TCA enzyme pool, 8 of the 48 parameters were adjustable. Simulation results are presented for verification of the model which successfully described the observed phenomena for the fermentations of S. cerevisiae strain AB103. 1 pYalphaEF-25. Also presented is a statistical analysis of the model's fit and model parameter sensitivity.

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

We have examined practical considerations associated with the use of a commercially available fluorescence probe for in situ measurements in bioreactors. The optical path length of the measurement was first determined and a flow cell subsequently designed. The environment (agitation/aeration rates) of the probe was found to have a significant influence on the measurement. These effects were eliminated by placing the probe in a recycle loop using the flow cell. Fluorescence measurement in the recycle loop was verified to be representative of the cell sample and to not affect cell metabolism.

Protein-secreting procaryotic host organisms are currently being sought as alternatives to Escherichia coli for recombinant processing. In this study we examined how manipulation of the cultivation conditions can enhance heterologous protein production by Streptomyces lividans. The recombinant S. lividans used in this study expressed and excreted a Flavobacterium enzyme capable of hydrolyzing organophosphates. Initial shake-flask studies demonstrated that supplementing Luria-Bertani medium with moderate amounts of glucose (30 g/l), led to improved enzyme production. In fermentor studies with controlled pH, a further twofold increase in production was observed when glucose was fed continuously as compared to batch cultivation. This improved production in the glucose-fed culture may be related to a reduced accumulation of acids. Continuous feeding of both glucose and tryptone led to a further sixfold increase in production. In addition to enhancing production 25-fold, the efficiency of enzyme production and the specific activity of the excreted enzyme were also improved by glucose and tryptone feeding. These results demonstrate that in addition to genetic manipulations, optimization of cultivation conditions can lead to significant improvements in the production of heterologous proteins from Streptomyces.

Recycle lines have been widely used in on-line analysis of fermentation processes. However, like most tools, many practical considerations can remain overlooked. Upon reflecting on this technique, some of these overlooked considerations have been studied. Because of the importance of a debubbler to a recycle line, an improved design was made and tested which can produce a bubble free high flow rate stream to reduce residence times. The effect of the recycle line on the culture growth, and DO was also investigated and found to be negligible.

Analysis of fermenter off-gas requires that the flowrate be accurately determined. We present an alternative to conventional methods which periodically spikes the off-gas flow with an accurately determined flow of argon. The resulting mol fraction of argon is determined by an existing mass spectrometer which in turn is used with a mol balance on argon to determine the total off-gas flowrate. For multiple fermenters this technique replaces expensive and maintenance intensive equipment with a single accurately controlled argon stream.

Expression kinetics of the human Epidermal Growth Factor (hEGF) from the alpha-factor prepro region in a 2-mum based plasmid was studied in Saccharomyces cerevisiae. Production of hEGF was highly medium de pendent as a chemically defined, nonenriched media had a significantly lower yield than did enriched media. Also cells grown on yeast nitrogen base without amino acids with casamino acids degraded the hEGF after cell growth as opposed to a yeast extract, peptone, and dextrose (YEPD) medium, which elicited no measurable extracellular proteolysis of the hEGF. alpha-factor directed production kinetics of hEGF on the YEPD medium were growth associated, secretion limitations and extracellular degradation were negligible, and the hEGF was nearly 100% selectively secreted. With sufficient agitation, shake flask experiments were representative of aerated controlled batch fermentations. No effect of high cell density was observed on cell growth or hEGF production kinetics. The hollow fiber bioreactor had no direct effect on the substrate or protein yields of S. cerevisiae, however the low oxygen transfer capacity of the membrane was not sufficient to support respiration.

The use of the yeast Saccharomyces cerevisiae has increased greatly over the past few years for the production of pharmaceuticals, specialty chemicals, and other commodities. One reason for this is the many advantages it offers for synthesis and secretion of recombinant DNA products. However, the growth characteristics of this yeast are quite complex and only recently has detailed analysis been available to provide additional insight into such phenomena as biphasic growth and catabolic repression. This paper summarizes and organizes current literature and also details the experimental behavior of on-line and off-line variables of S. cerevisiae in glucose-limited-batch, fed- batch, and hollow-fiber bioreactors. The observed behavior is interpreted using knowledge of yeast catabolism biochemistry and physiology. Special focus is placed on the interpretation of off-gas behavior with respect to TCA enzyme activity and the saturation level of oxygen utilization capacity.

The Nucleogen DEAE 4000-10 high-performance liquid chromatography (HPLC) column was shown to isolate tRNA, rRNA, and DNA from crude cell lysates in the presence of a denaturant. Chromosomal DNA and plasmid DNA coeluted and retention was independent of size. An HPLC technique for plasmid copy number determination was then developed, verified, and optimized. The assay was successfully applied to a recombinant yeast system. Similar results were obtained earlier for recombinant Escherichia coli . The copy number can be calculated from the ratio of plasmid DNA to rRNA, then calculated to a per-cell basis using literature and measured constants. Chromosomal DNA coeluted with plasmid DNA and so was removed prior to analysis. For recombinant systems were the rRNA content is not constant, the plasmid area along with cell concentration can be used to calculate plasmid copy number. Nucleic acids′ responses were linear, and plasmid copy number results using peak ratios had a low percent standard deviation. Errors increased when the absolute plasmid area was used to calculate plasmid copy number.